OCCURRENCE OF STAPHYLOCOCCUS AUREUS IN FAST FOOD WITH SPECIAL REFERENCE TO ITS ENTEROTOXIGENICITY

Animal Health Research Institute.

Port Said Laboratory, Food Hygiene,

Occurrence of Staphylococcus aureus in fast food with special reference to its enterotoxigenicity

(With 4 Tables)

By

Amany M. Shalaby and Eman M.S. Zaki*

* Bacterial Toxins Unit, Animal Health Research Institute, Dokki

(Received at 2/2/2008)

تواجد الميکروب المکور العنقودى الذهبى فى الوجبات السريعة مع أشارة خاصة لسميتة المعوية

 أمانى محمود شلبى ،  أيمان محمد شوقى زکى

أجريت هذه الدراسة لمعرفة مدى تلوث الوجبات السريعة (ساندويتشات) المجهزة للأکل من منتجات اللحوم (شاورمة – سجق – برجر) والمجمعة من محلات وبائعى الوجبات السريعة بمدينة بورسعيد بميکروب المکور العنقودى الذهبى وکذلک للوقوف على معرفة مدى إفراز العترات المعزولة للسموم المعوية المسببة للتسمم الغذائى. ولقد شملت الدراسة إجراء  الفحص البکتيريولوجى لعدد 75 عينة حيث أفضت الى وجود الميکروب المکور العنقودى الذهبى بنسب 32%، 44%، 36% وکذا کان العد البکتيرى للميکروب 9.8 × ²10 ، 1.2 × ³10 ، 8.3 × ²10 فى کل من الشاورمة والسجق والبرجر على التوالى. وتم تصنيف العترات المعزولة لمدى إفرازها للسموم ولوحظ أن نوع السم D , A الأکثر وجوداً فى العينات المجمعة. ولقد نوقشت الأهمية الصحية للميکروب وسمومه ووضعت التوصيات اللازمة لسلامة المستهلک والمنتج.

SUMMARY

A total of 75 random samples of fast food (ready-to-eat meat sandwiches) represented as 25 each of shawarma, sausage and beef burger were collected from various fast food restaurants and vendors on the street in Port Said city. S. aureus could be detected in 32, 44 and 36% of the analyzed samples with a mean value of 9.8 x 10² ± 0.12 x 10²; 1.2 x 10³ ± 0.24 x 10² and 8.3 x 10² ± 0.09 x 10² CFU/g shawarma, sausage and beef burger, respectively. Out of 8 strains obtained from shawarma, only 3 were enterotoxigenic belonging to type A enterotoxin. Five S. aureus were enterotoxigenic obtained from sausage samples, 2 of them were type A and the other 3 strains were enterotoxins AD. Furthermore, 4 strains identified as 2 type AB producers and 2 type ABCD producers were isolated from beef burger samples. The results showed that enterotoxin A (SEA) was the most frequently in all the examined ready-to-eat-sandwiches followed by enterotoxin type D (SED), indicating that S. aureus had a potential public health significance in fast food.

Key words: Fast food, shawarma, sausage, beef burger, staph aureus

INTRODUCTION

In Egypt as in many other African countries, fast foods are commonly vended on streets and at market sites in urban area where there is constant and heavy movement of people. Although there are no readily obtainable epidemiological data about the risks of food borne diseases resulting from these foods, the laboratory data show that street vended foods frequently have high microbial populations and occasionally high population of pathogenic bacteria (Bryan, 1988 & 1992).

Nowadays, meat products consumed as sandwiches of shawarma, sausage, beef burger, etc. are commonly prepared and sold by many restaurants which are widely distributed all over the country (Take- away). Staphylococcus aureus is one of the most important microorganisms which can contaminate or recontaminate cooked foods via workers hands, equipments or utensils (Bryan, 1978 & 1988). The primary reservoirs of S. aureus are human skin and mucosa especially the nasopharyngeal cavity (Bystron et al., 2005).

Staphylococcal food poisoning resulting from the growth of enterotoxigenic strains of S. aureus in food leading to the production of enterotoxins which is considered one of the major causes of food borne disease all over the world (Minor and Marth, 1972).

Enterotoxins are groups of single chain protein (poly peptides) with molecular weight 28.000- 35.000 Daltons resistant to high temperature (heat stable) and proteolytic enzymes. The enterotoxigenic strains of S. aureus produce several types of enterotoxins (A, B, C, D and E) which can cause symptoms of intoxications such as vomiting, diarhoea and abdominal cramping (Korpysa et al., 2005). Enterotoxin A (SEA) is responsible for a majority of staphylococcal food poisoning whereas enterotoxin B (SEB) is rarely involved (Robbins et al., 1974). In addition most outbreaks as recorded by Halpin-Dohnalek and Marth (1989) resulted from the combined effect of contamination of the food with S. aureus often through unsanitary handling, and holding the food at the wrong temperature thus allowing growth and synthesis of enterotoxins. However, the enterotoxication generally is not lethal and the elderly are more susceptible than younger individuals. The amount of enterotoxin necessary to cause intoxication is very small about 94- 184 ng (Erol and Iseri, 2004).

Therefore, the purpose of such investigation is to record the occurrence of S. aureus in some different fast meat food and its ability to produce enterotoxins.

MATERIALS and METHODS

1. Collection of samples:

A total of 75 random samples of fast meat food were purchased from different fast food restaurants and vendors on street in Port Said city. The samples were represented as 25 each of ready-to-eat shawarma, sausages and beef burger sandwiches. The samples were directly transferred to the laboratory in an ice box under hygienic conditions without delay to be examined bacteriologically.

2. Preparation of the samples:

According to the technique recommended by APHA (1992), ten grams of each meat product sample only without bread were put into the stomacher bag, to which 90 ml of sterile physiological saline (0.9 %) were aseptically added to provide dilution of 1/10, then the content of the bag was stomached for 60 sec, using the stomacher, then 10 fold serial dilutions were prepared.

3. Determination of S. aureus count:

Staphylococcus aureus count/g was determined using surface plating technique according to FAO (1992). 0.1 ml from each of the previously prepared decimal dilutions was transferred onto duplicate plates of Baird- parker media, supplemented with egg yolk tellurite and incubated at 37°C for 24- 48 hrs. Then the black and shiny colonies greater than 1 mm in diameter with narrow white margin surrounded by clear zone extending into opaque medium were counted and recorded.

4. Isolation and Identification of S. aureus (FAO, 1992):

Suspected colonies of S. aureus were subcultured on slants of brain heart infusion agar (Oxoid) (FAO, 1992) and incubated at 37°C for 24 hrs. Isolated purified strains were identified microscopically and biochemically for confirmation according to Flandrois and Carret(1981), Marchant and Parker (1983) and Bailley and Scott (1990).

5. Production and detection of enterotoxins from the isolated strains of S. aureus:

a- Production of staphylococcal enterotoxins from the isolated strains:

            The isolated strains of S. aureus were examined for their ability to produce enterotoxins using Sac culture method (Donnelly et al., 1967).

b- Detection and typing of enterotoxins:

            According to Oda et al. (1979) and Shingaki et al. (1981), detection and typing of enterotoxins were done using serological test by reversed passive latex agglutination technique using Oxoid SET- RPLA (kit used for the detection of staphylococcal enterotoxins A, B, C        and D).

6. Detection of staphylococcal enterotoxins in the food samples (Direct extraction):

a- Toxin extraction: the steps including preparation of the food samples and toxin extraction from the food itself described by the method recommended by Park and Szabo (1986) were followed.

b- Enterotoxin detection: as mentioned previously methods recommended by Oda et al. (1979) and Shingaki et al. (1981)was followed using SET- RPLA kit in which antibody- coated latex particles are visibly agglutinated in the presence of SET (Staphylococcal Enterotoxins). It is commercially available in kit form Oxoid Ltd (Basing stoke, Hampshire, UK).

RESULTS

Table 1: Incidence of S. aureus in the examined fast food samples.

      * Non significant

Table 2: Statistical results of S. aureus count /g of the examined fast food samples.

Mean of the positive samples.

# Significant at P < 0.05 using ANOVA test.

a, b unsignificant difference between similar litter at P < 0.05 using Duncan Multiple Range Test.

Table 3: Frequency distribution of theexamined fast food samples based on their S. aureus count.

Table 4: Incidence of enterotoxigenic S. aureus and types of its enterotoxins.

* Non significant

DISCUSSION

            The results in Table 1 revealed that S. aureus was present in    32, 44 and 36% of shawarma, sausage and beef burger sandwiches, statistically there are no significant difference between the three ready to eat meat products.

It is obvious from such incidence that the level of contamination by S. aureus in the examined samples was relatively high, since these fast food are ready-to-eat in the form of sandwiches without any further heat processing. In this respect Wieneke (1974) isolated S. aureus      (32-36%) from cooked foods including meat and she added that S. aureus strains isolated from human resembled those from cooked foods. While, Halpin-Dohnalek and Marth (1989) stated that outbreaks of S. aureus food poisoning are most often associated with processed red meats and most outbreaks resulted from the combined effects of contamination of the food with S. aureus, often through unsanitary handling and holding the food at the wrong temperature thus allowing growth and synthesis of enterotoxins by the pathogen. Alkanahl and Gasim (1993) found that cooked foods (both unheated and reheated) were associated with more incidents of food borne illness cases (56%) than uncooked foods (20%), S. aureus was found in both cooks and suspect foods. It was also found high % of the cooks associated with food borne incidents had no health certificates.

Regarding the incidence of S. aureus recorded in Table 1, the obtained results were less than those obtained by El-Daly (1983); El-Sherbeeny et al. (1985); Alkanahl and Gasim (1993); Kumar et al. (2001) and higher than those obtained by Soriano et al. (2000), Little et al. (2001); Firinu et al. (2003); Shabana and Ouf (2003). Bystron (2005) and Manfreda et al., (2005) recorded similar results. On the other hand Hassan (1991), Hegazy (1999) and Ebraheem (2001) can not count S. aureus from ready to eat meat sandwiches. Sokari and Anozie (1990) suggested that the high level of contamination with S. aureus resulted from cross-contamination reflecting excessive hand contact with food stuffs.

            As shown in Table 2 that illustrated the mean values of S. aureus count in the examined fast meat samples were 9.8 X 10² ± 0.12 X 10², 1.2 X 10³ ± 0.24 X 10² and 8.3 X 10² ± 0.09 X 10² CFU/g in shawarma, sausage and beef burger, respectively.

Statistically, there are significant differences between shawarma, beef burger and sausage, the later is higher in its S. aureus count than the others. These results are nearly agree with El-Daly (1983); Hoshyar, et al. (1984). Ayaz, et al. (1985) and Morshdy, et al. (1986). Lower findings achieved by Soliman (1988), Nassar (1988), Refaie and Moustafa (1990) and Daif (1996), while more than those obtained by El-Sherbeeny et al. (1985), Fathi (1988) and Little et al. (2001). In this respect, Frazier and Westhaff (1978) reported that the growth of S. aureus in cooked or sterilized food is better than in raw foods if contamination occurs after manufacture and also they reported that the risk of S. aureus in burger is relatively low compared to the volume of burger eaten, this may be due to burger is usually cooks well.

Outbreaks could be resulted when processed or cooked meat has been improperly handled and mistreated in food processing plants and/or catering and food service establishments (ICMSF, 1980). Zein El-Abdin et al. (1992) reported that the increase in the bacterial counts of meat meals during distribution in aluminum dishes could be attributed to the holding temperature.

The results in Table 3 showed that the highest frequency distribution for shawarma and beef burger lies within the range of      10²-10³ S. aureus/g., while that of sausage samples was within 10³-10⁴. Wieneke et al. (1993) recorded that meat and meat products were the vehicles of 75% of incidents of food poisoning with ham and chickens implicated, in the United Kingdom, (1969-90) and the level of S. aureus present in food ranged from no viable cells detected to 1.5 X 10¹⁰ C.F.U./g. with a median of 3 X 10⁷ C.F.U./g. Wamola (1992) stated that the handling and preparation of food when in correct manner can reduce the level of microbial contamination thereby enhancing shelf life and he added that, certain foods carry a higher risk of microbial contamination than others. Little et al. (2001) isolated S. aureus from ready-to-eat burger with a level of 10² CFU/g, while Elmal et al. (2005) could detect S. aureus in fast beef doner kebabs in counts <10² to 10⁴ CFU/gm. In this respect Fernandez et al. (2006) evaluated the effect of storage time and temperature on the microbiological quality of meat burger. They determined S. aureus in the burger samples and stated that fast food restaurant must follow the international standards.

As shown in Table 4 the results illustrated that the incidence of enterotoxins produced by S. aureus in shawarma was 37.5%, where       3 strains belonged to type A. In sausage, 5 (45.5%) of the recorded strains were enterotoxigenic where 2 of them were type A enterotoxin and the other 3 strains were enterotoxins AD. Four enterotoxigenic strains were obtained from beef burger samples (44.5%) and were identified as 2 type AB and 2 type ABCD staphylococcal enterotoxins. These results agree with those obtained by Adesiyun (1984) who detected enterotoxins from ready-to-eat beef S. aureus isolates (43.3%). The author stated that beef isolates of S. aureus were the most frequently toxic than other food products and may pose a health hazard with high incidence of enterotoxin type D. Bergdoll (1989) reported that S. aureus causes food poisoning by the production of one or more heat stable extracellular toxins, responsible for the symptoms of the disease, time of onset and severity of symptoms depend on the amount of toxin consumed and individual susceptibility. The enterotoxins produced at detectable levels (> 0.1 ng/gm) in foods occurs only when growth reaches approximately 10⁶ CFU/gm (Robbins et al., 1974 and Evenson et al., 1988).

The summarized results recorded in Table 4 nearly agree with those obtained by Adesiyun (1984); Rosec et al. (1997); Holeckova et al. (2002); Firinu et al. (2003) and Bystron (2005). Lower findings were achieved by Kumar et al. (2001) and Manfreda et al. (2005). It was found also that enterotoxin type A (SEA) is the most frequently followed by enterotoxin type D (SED) such results go parallel with the results of Minor and Marth (1972); Helena-Lopes et al. (1993) and Erol and Iseri (2004), they reported that staphylococcal enterotoxins A (SEA) and      D (SED) being the most responsible for the majority of staphylococcus food poisoning. However, there is no relation between the enterotoxigenicity and the other biochemical charcateries (Bystron et al., 2002). On the other hand, the direct extraction of staphylococcal enterotoxins from the food samples revealed negative results (not detectable). Halpin-Dohnalek and Marth (1989) stated that enterotoxins can cause food poisoning are produced by about one-third (1/3) of the coagulase positive strains of S. aureus and the production of enterotoxigns is affected by the nutritional quality and pH of the substrate, temp., atmosphere, sodium chloride, water activity, other chemicals and competing microorganisms. It is benefit to mention that the reversed passive Latex agglutination assay (RPLA) for staphylococcal enterotoxins detection is a method based on the specific antibodies directed to the enterotoxins and characterized by its accuracy and easy to use. Bergdoll (1990) stated that the detection of S. aureus enterotoxins in food requires much more sensitive methods than those required for the determination of enterotoxigenicity of strains, and the quantity of enterotoxin present in foods involved in food poisoning outbreaks vary from less than 1 ng/gm to greater than 1 μg/gm food.

Considering the importance and public health hazard of S. aureus organism recovered from fast food (ready-to-eat meat sandwiches), Longree and Blacker (1971) reported that preparing and serving food to the public is a very important obligation that can only be fulfilled if every one in the establishment understand food hygiene, applying sanitary measures at every stage of the operation. Furthermore, ICMSF (1988) stated that cooked meat should not be touched by hands or by equipments that have come in contact with raw meat equipments that have come in contact with raw meat, raw products should be separated from cooked products to avoid cross-contamination.

To safe the ready-to-eat sandwiches sold in fast food services it must be focus on prevention of contamination and multiplication of microbes and production of toxins. Food should not be prepared long in advance of consumption (Bryan et al., 1992). Cooking usually give time temperature exposures that would have been lethal for vegetative form of food borne pathogens. On the other hand, holding of food provide time temperature exposures conductive to microbial growth, particularly in food helds overnight and large populations of aerobic organisms including S. aureus and others were recovered from these food. So, time temperature had variable effect of killing the microorganism but heat stable toxins still not affected (Jermini et al., 1997; Pepe et al., 2006).

In conclusion, it can be achieved from the obtained data that fast meat products (sandwiches) have the potential to cause staphylococcal intoxication to consumers. So, the rules of health agencies must reach to all workers in such field especially street vendors and fast food takeaway restaurants besides safety programs for safe food preparation drawn by WHO (1989) should be followed and effective preventive measures must be authorized and applied to safe the consumer health.

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